ProgrramDiriercetcotro/Pr/rPinrcinipcaipllnavl eInsvtiegastoigra(Ltoarst(,LFaisrstt,,FMiridsdt,leM):idMdAleD):IMRADJIURA, JMU, RMTUYRTVY3VR35R65A6 IA7l 733996-015S1I .rn virulence factor. with membranes and the ability of DnaA to initiate replication depends, promote NRP state in vivo are largely unknown. Our proposal focuses on DNA replication. interactions with active, but in a non-replicative persistent (NRP) state. growth. The genetic elements responsible for regulation of Mtb DnaA protein initiates this process at a unique site on the genome called oriC. DnaA associates multiplication of organisms and this process is believed to be regulated during intracellular maintains an active replicative state with increase in bacterial burden, membrane lipids, possibly modulated by LysX, a hitherto unrecognized Z replication and or the factors that in part, on its ABSTRACT Tuberculosis remains a major global public health crisis despite being a curable disease. One Characteristic feature of Mycobacterium tuberculosis (Mtb),, the causative agent of tuberculosiis, is its slow growth. Mtb employs multiple regulatory networks for maintaining its optimal growth in vivo. The consequences of Mtb-host interactions believed to dictate whether the pathogen maintains an active replicative state with increase in bacterial burden, or remains metabolicaly active, but in a non-replicative persistent (NRP) state. DNA replication is essential for the multiplication of organisms and this process is believed to be regulated during intracellular growth. The genetic elements responsible for regulation of Mtb replication and or the factors that promote NRP state in vivo are largely unknown. Our proposal focuses on DNA replication. DnaA protein initiates this process at a unique site on the genome called oriC. DnaA associates with membranes and the ability of DnaA to initiate replication depends, in part, on its interactions with membrane lipids, possibly modulated by LysX, a hitherto unrecognized virulence factor. [unreadable]-' gin' =r0 --1 --o L'- 6m_ E V-2 3-=v >.C turn 'n. Vii' -.0 Q(. these experiments will define how the DNA replication process in Mtb is regulated, hence will production of lysinylated polar lipids (P0L), thereby influences membrane fluidity, hence DnaA improve our understanding of proliferation of Mtb in vivo. membrane fluidity result in replication arrest and induction of Mtb persistence. It is hoped that activity. A long-term goal of these studies is to understand how the LysX regulated changes in 'L- We propose that in response to infection one regulatory pathway mediated by the LysX protein regulates DnaA activity, hence replication initiation. A consequence of this regulation determines whether DNA replication wil continue resulting in active multiplication, or stall/shut-down contributing to NRP state. We wil dissect this pathway in an attempt to define the roles of LysX on DnaA activity and replication initiation during intracellular growth. Specificaly, we wil examine if l/ysX is an unrecognized virulence factor,, and if it regulates the production of Iysinylated polar lipids (PoL), thereby influences membrane fluidity, hence DnaA activity. A long-term goal of these studies is to understand how the LysX regulated changes in membrane fluidity result in replication arrest and induction of Mtb persistence. It is hoped that these experiments will define how the DNA replication process in Mtb is regulated, hence will improve our understanding of proliferation of Mtb in vivo. 00m 0-.(l ,-. [unreadable]=Z ESL m.=o can NC: C.-taco ='o (n>-CSoEZ 0--(a ML)